Alexander V. Vorotnikov

Activation of p38 MAP-Kinase and Caldesmon Phosphorylation Are Essential for Urokinase-Induced Human Smooth Muscle Cell Migration

Abstract We have explored intracellular pathways involved in the urokinase type plasminogen activator (urokinase or uPA)stimulated migration of human airway smooth muscle cells (hAWSMC). Using a set of uPA mutants we found that protease activity, growth factorlike and kringle domains of uPA differentially contribute to activation of p42/p44erk1,2 and p38 MAPkinases. Consistent with our earlier data [Mukhina et al., J. Biol. Chem. 275 (2000), 16450 16458], the kringle domain of uPA was sufficient and required to stimulate cell motility. Here we report that uPA mutants containing the kringle domain specifically activate the p38 MAPkinase pathway and actomyosin by increasing phosphorylation of the critical Ser-19 on the myosin regulatory light chain and MAPkinase sites of the actinassociated regulatory protein caldesmon. While pharmacological inhibition of p38 MAPkinase activation did not affect myosin light chain phosphorylation, it blocked the increase in caldesmon phosphorylation and uPAstimulated migration of hAWSMC on a collagencoated surface. We conclude that activation of p38 MAPkinase and downstream phosphorylation of nonmuscle caldesmon is essential for urokinasestimulated smooth muscle cell migration.

Sites of Interaction between Kinase-related Protein and Smooth Muscle Myosin

Kinase-related protein, also known as KRP or telokin, is an independently expressed protein product derived from a gene within the gene for myosin light chain kinase (MLCK). KRP binds to unphosphorylated smooth muscle myosin filaments and stabilizes them against ATP-induced depolymerization in vitro. KRP competes with MLCK for binding to myosin, suggesting that both proteins bind to myosin by the KRP domain (Shirinsky, V. P., Vorotnikov, A. V., Birukov, K. G., Nanaev, A. K., Collinge, M., Lukas, T. J., Sellers, J. R., and Watterson, D. M. (1993)J. Biol. Chem. 268, 16578–16583). In this study, we investigated which regions of myosin and KRP interact in vitro. Using cosedimentation assays, we determined that KRP binds to unphosphorylated myosin with a stoichiometry of 1 mol of KRP/1 mol of myosin and an affinity of 5.5 μm. KRP slows the rate of proteolytic cleavage of the head-tail junction of heavy meromyosin by papain and chymotrypsin, suggesting it is binding to this region of myosin. In addition, competition experiments, using soluble headless fragments of nonmuscle myosin, confirmed that KRP interacts with the regulatory light chain binding region of myosin. The regions important for KRP’s binding to myosin were investigated using bacterially expressed KRP truncation mutants. We determined that the acid-rich sequence between Gly138 and Asp151 of KRP is required for high affinity myosin binding, and that the amino terminus and β-barrel regions weakly interact with myosin. All KRP truncations, at concentrations comparable to theirK D values, exhibited some stabilization of myosin filaments against ATP depolymerization in vitro, suggesting that KRP’s ability to stabilize myosin filaments is commensurate with its myosin binding affinity. KRP weakened the K m but not the V max of phosphorylation of myosin by MLCK, demonstrating that bound KRP does not prevent MLCK from activating myosin.

Chemotaxis: Movement, direction, control

This review focuses on basic principles of motility in different cell types, formation of the specific cell structures that enable directed migration, and how external signals are transduced into cells and coupled to the motile machinery. Feedback mechanisms and their potential role in maintenance of internal chemotactic gradients and persistence of directed migration are highlighted.

Phosphorylation of aorta caldesmon by endogeneous proteolytic fragments of protein kinase C

SummaryEndogenous caldesmon kinase activity in sheep aorta smooth muscle was purified and characterized. The enzyme was identified as a proteolytic fragment of protein kinase C by cross-reactivity with anti-protein kinase C antibodies, autophosphorylation, substrate specificity and the primary structure of the sites of phosphorylation on caldesmon. The enzyme phosphorylated aorta caldesmon both in native thin filaments and in the isolated state. Up to 2.9 mols of phosphate per mol of caldesmon were transferred. Prolonged incubation of caldesmon with the kinase resulted in phosphorylation of Ser-127, Ser-587, Ser-600, Ser-657, Ser-686, and Ser-726 (numbering corresponds to chicken gizzard caldesmon sequence). Ser-600 and Ser-587 were the major sites of phosphorylation containing more than 30% of phosphate transferred. Phosphorylation did not significantly affect the interaction of caldesmon with Ca2+-calmodulin. However, phosphorylation of both intact caldesmon and of its C-terminal fragment (658C), containing residues 658–756, significantly decreased their ability to inhibit acto-heavy meromyosin ATPase. This seems to be partially due to a decrease in the binding of caldesmon and 658C to actin-tropomyosin and partly due to an uncoupling of the binding-inhibition relationship.

Phosphorylation of kinase-related protein (telokin) in tonic and phasic smooth muscles

KRP (telokin), an independently expressed C-terminal myosin-binding domain of smooth muscle myosin light chain kinase (MLCK), has been reported to have two related functions. First, KRP stabilizes myosin filaments (Shirinsky et al., 1993, J. Biol. Chem. 268, 16578–16583) in the presence of ATP. Secondly, KRP can modulate the level of myosin light chain phosphorylation. In this latter role, multiple mechanisms have been suggested. One hypothesis is that light chain phosphorylation is diminished by the direct competition of KRP and MLCK for myosin, resulting in a loss of contraction. Alternatively, KRP, through an unidentified mechanism, accelerates myosin light chain dephosphorylation in a manner possibly enhanced by KRP phosphorylation. Here, we demonstrate that KRP is a major phosphoprotein in smooth muscle, and use a comparative approach to investigate how its phosphorylation correlates with sustained contraction and forskolin-induced relaxation. Forskolin relaxation of precontracted artery strips caused little increase in KRP phosphorylation, while treatment with phorbol ester increased the level of KRP phosphorylation without a subsequent change in contractility. Although phorbol ester does not induce contraction of phasic tissues, the level of KRP phosphorylation is increased. Phosphopeptide maps of KRP from both tissues revealed multiple sites of phosphorylation within the N-terminal region of KRP. Phosphopeptide maps of KRP from gizzard were more complex than those for KRP from artery consistent with heterogeneity at the amino terminus and/or additional sites. We discovered through analysis of KRP phosphorylation in vitro that Ser12, Ser18 and Ser15 are phosphorylated by cAMP-dependent protein kinase, mitogen-activated protein (MAP) kinase and glycogen synthase kinase 3 (GSK3), respectively. Phosphorylation by GSK3 was dependent upon prephosphorylation by MAP kinase. This appears to be the first report of conditional or hierarchical phosphorylation of KRP. Peptides consistent with such multiple phosphorylations were found on the in vivo phosphopeptide maps of avian KRP. Collectively, the available data indicate that there is a complex relationship between the in vivo phosphorylation states of KRP and its effects on relaxation in smooth muscle.

Urokinase induces matrix metalloproteinase-9/ gelatinase B expression in THP-1 monocytes via ERK1/2 and cytosolic phospholipase A2 activation and eicosanoid production

Objective: Urokinase-type plasminogen activator (uPA) regulates cell migration and invasion by pericellular proteolysis and signal transduction events. We characterized the mechanisms by which uPA regulates matrix metalloproteinase-9 (MMP9) function in THP-1 monocytes. Methods and Results: In THP-1 monocytes, MMP9 production induced by urokinase was completely inhibited by the ERK1/2 inhibitor, PD98059, but not by the p38 mitogen-activated protein kinase inhibitor, SB202190. A dominant negative MEK1 adenovirus also blocked MMP9 expression. The effect of urokinase was completely suppressed by genistein and by herbimycin A indicating that tyrosine kinase(s) are required for MMP9 production. Bisindolylmaleimide, a protein kinase C (PKC) inhibitor, did not decrease MMP9 expression suggesting that PKC activation is not required. Key roles for cytosolic phospholipase A2 (PLA2) and eicosanoid production were shown by complete inhibition with methyl arachidonyl fluorophosphonate (an inhibitor of cytosolic PLA2), and indomethacin (a cyclooxygenase inhibitor), with no effect of monoalide, a secretory PLA2 inhibitor. uPA stimulated phosphorylation of cytosolic PLA2. Conclusions: Induction of MMP9 by uPA in THP-1 monocytes is via a pathway involving MEK1-ERK1/2-mediated activation of cytosolic PLA2 and eicosanoid generation. These data suggest important roles for eicosanoids in monocyte migration induced by uPA and MMP9.

Evidence against the regulation of caldesmon inhibitory activity by p42/p44erk mitogen-activated protein kinase in vitro and demonstration of another caldesmon kinase in intact gizzard smooth muscle

The effect of direct phosphorylation by recombinant p44erk1 mitogen‐activated protein kinase on the inhibitory activity of caldesmon and its C‐terminal fragment H1 was studied in vitro. Neither inhibition of actin‐tropomyosin activated ATPase of heavy meromyosin by caldesmon or H1, nor inhibition of the actin‐tropomyosin motility over heavy meromyosin by H1 was significantly affected by the phosphorylation while only a moderate effect on the actin‐activated component of heavy meromyosin ATPase inhibition was observed. Phosphopeptide mapping of caldesmon immunoprecipitated from [32P]PO4‐labelled intact gizzard strips revealed that it is predominantly phosphorylated at mitogen‐activated protein kinase sites in unstimulated tissue and that it is stimulated for 1 h with phorbol 12,13‐dibutyrate. We find that phorbol 12,13‐dibutyrate also induces a transitory phosphorylation of caldesmon peaking at 15 min after addition and this phosphorylation is not attributed to mitogen‐activated protein kinase, protein kinase C, Ca2+/calmodulin‐dependent kinase II or casein kinase II. We suggest that a yet unidentified kinase, rather than mitogen‐activated protein kinase, may be involved in regulation of the caldesmon function in vivo.

Fibulin-5 binds urokinase-type plasminogen activator and mediates urokinase-stimulated β1-integrin-dependent cell migration.

uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility.

Phosphorylation of the minimal inhibitory region at the C-terminus of caldesmon alters its structural and actin binding properties

Caldesmon is an inhibitory protein believed to be involved in the regulation of thin filament activity in smooth muscles and is a major cytoplasmic substrate for MAP kinase. NMR spectroscopy shows that the actin binding properties of the minimal inhibitory region of caldesmon, residues 750-779, alter upon MAP kinase phosphorylation of Ser-759, a residue not involved in actin binding. This phosphorylation leads to markedly diminished actin affinity as a result of the loss of interaction at one of the two sites that bind to F-actin. The structural basis for the altered interaction is identified from the observation that phosphorylation destabilises a turn segment linking the two actin binding sites and thereby results in the randomisation of their relative disposition. This modulatory influence of Ser-759 phosphorylation is not merely a function of the bulkiness of the covalent modification since the stability of the turn region is observed to be sensitive to the ionisation state of the phosphate group. The data are discussed in the context of the inhibitory association of the C-terminal domain of caldesmon with F-actin.

Identification of the site phosphorylated by casein kinase II in smooth muscle caldesmon

Phosphorylation of avian gizzard caldesmon by casein kinase II was investigated. The enzyme incorporates about 1 mol of phosphate per mol of caldesmon. All sites of phosphorylation are located in short chymotryptic peptides with M r, 25–27 kDa or in the short N‐terminal peptide formed after cleavage of chicken gizzard caldesmon at Cys153. The primary structure of the tryptic peptide containing the main site of duck gizzard caldesmon phosphorylation is S‐E‐V‐N‐A‐Q‐N‐X‐V‐A‐E‐D‐E‐T‐K. where X is an unidentified residue. presumed to be phosphorserine. Thus, Ser73 is the main site phosphorylated by casein kinase II in avian gizzard caldesmon.